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单分子磁镊技术在生理相关力和时间尺度下探测单个蛋白质的平衡动力学。

Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.

机构信息

Single Molecule Mechanobiology Laboratory, The Francis Crick Institute, London, UK.

Department of Physics, Randall Centre for Cell and Molecular Biophysics, Centre for the Physical Science of Life and London Centre for Nanotechnology, King's College London, London, UK.

出版信息

Nat Protoc. 2024 Jun;19(6):1779-1806. doi: 10.1038/s41596-024-00965-5. Epub 2024 Mar 11.

DOI:10.1038/s41596-024-00965-5
PMID:38467905
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7616092/
Abstract

The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis.

摘要

蛋白质的可还原折叠和重折叠是组织弹性和信号传导的一种调节机制，细胞可通过这种机制感知和适应细胞外和细胞内的机械力。然而，这些蛋白质中的大多数表现出较低的机械稳定性，这给在力作用下对其构象动力学进行特征描述带来了技术挑战。本文详细介绍了使用超稳定磁镊进行单蛋白纳米力学实验的分步说明，该实验可在生理相关的低力和生物相关的时间尺度下测量单个蛋白质的平衡构象动力学。我们报告了决定磁镊仪器功能的基本原理，综述了蛋白质设计策略和流体室的制备，并详细介绍了在力作用下获取和分析单个蛋白质的展开和重折叠轨迹的过程。这项技术增加了单分子纳米力学技术的工具箱，对于那些对参与机械感应和机械转导的蛋白质感兴趣的人来说尤其有吸引力。整个过程需要 4 天完成，加上蛋白质克隆和生产还需要另外 6 天，这需要分子生物学、表面化学和数据分析方面的基本知识。

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